CN114908158A

CN114908158A - Application of CDK1 in diagnosis and treatment of advanced gastrointestinal stromal tumor

Info

Publication number: CN114908158A
Application number: CN202110172517.9A
Authority: CN
Inventors: 王跃祥; 芦晓晶
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2022-08-16
Anticipated expiration: 2041-02-08

Abstract

The present invention relates to the use of CDK1 in the diagnosis and treatment of advanced gastrointestinal stromal tumors. Specifically, the present invention provides a use of CDK1 gene, mRNA, cDNA, or protein or a detection reagent thereof as (i) a marker for detecting advanced gastrointestinal stromal tumors; and/or (ii) for the preparation of a diagnostic reagent or kit for the detection of advanced gastrointestinal stromal tumors, and the inhibitor of the CDK1 gene or protein thereof of the present invention is optionally in combination with a tyrosine kinase inhibitor, and/or optionally other drugs for the prevention and/or treatment of advanced gastrointestinal stromal tumors, and has a significant synergistic effect on the treatment of advanced gastrointestinal stromal tumors.

Description

Application of CDK1 in diagnosis and treatment of advanced gastrointestinal stromal tumor

Technical Field

The present invention relates to the fields of oncology and diagnostics. More specifically, the invention relates to the use of CDK1 in the diagnosis and treatment of advanced gastrointestinal stromal tumors.

Background

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the gastrointestinal tract. The gastrointestinal stromal tumor is divided into primary and metastatic gastrointestinal stromal tumor according to the existence of metastasis, the primary gastrointestinal stromal tumor is divided into low-risk, medium-risk and high-risk gastrointestinal stromal tumor according to pathological indexes (tumor size, mitosis number per high-powered visual field, anatomical position and the like), and the high-risk stromal tumor refers to high risk level of metastasis and recurrence. High-risk gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors are collectively referred to as advanced gastrointestinal stromal tumors. Clinically, the treatment principles and protocols for interstitial tumors vary in different risk levels. After receiving the standard treatment scheme, part of the intermediate-risk or low-risk gastrointestinal stromal tumors still have poor curative effect (reflected by tumor recurrence or metastasis). There is therefore a great need in the art to develop targets for gastrointestinal stromal tumors that have value in assessing patient risk levels.

In addition, advanced gastrointestinal stromal tumors are often misdiagnosed as other gastrointestinal tumors (e.g., gastrointestinal smooth muscle tumors, gastrointestinal schwannoma, etc.), so there is a need in the art to develop targets for differential diagnosis of advanced gastrointestinal stromal tumors.

And currently, for patients with gastrointestinal stromal tumors receiving targeted therapy, the majority of patients develop resistance.

Therefore, there is an urgent need in the art to develop new targets with diagnostic and therapeutic effects and new methods to overcome drug resistance and sensitivity.

Disclosure of Invention

The aim of the invention is to provide new targets for diagnosis and therapy and new methods for overcoming resistance and sensitivity.

The invention provides in a first aspect the use of a CDK1 gene, mRNA, cDNA, or protein, or a detection reagent therefor, (i) as a marker for the detection of advanced gastrointestinal stromal tumors; and/or (ii) for preparing a diagnostic reagent or kit for detecting advanced gastrointestinal stromal tumors.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

In another preferred embodiment, the CDK1 gene, mRNA, cDNA, or protein is derived from a mammal, preferably from a rodent (e.g., mouse, rat), primate, and human, more preferably from a patient diagnosed with advanced gastrointestinal stromal tumors.

In another preferred embodiment, the CDK1 gene, mRNA, cDNA, or protein is derived from a patient having advanced gastrointestinal stromal tumors.

In another preferred embodiment, the CDK1 gene has the accession number NG _ 029877.1.

In another preferred embodiment, the CDK1 mRNA has the accession number NM _ 001786.

In another preferred embodiment, the CDK1 protein has the accession number NP _ 001777.

In another preferred embodiment, the test is a tissue sample test.

In another preferred embodiment, the detection comprises immunohistochemistry, immunoblotting, co-immunoprecipitation and fluorescent quantitative PCR detection.

In another preferred embodiment, the detection is of an early stage gastrointestinal stromal tumor tissue, or a late stage gastrointestinal stromal tumor tissue sample.

In another preferred embodiment, the detection reagent comprises an antibody specific for CDK1, a binding molecule specific for CDK1, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the detection reagent is selected from the group consisting of: antibodies, primers, probes, sequencing libraries, nucleic acid chips (e.g., DNA chips), protein chips, or combinations thereof.

In another preferred embodiment, the CDK1 protein or specific antibody or specific binding molecule thereof is coupled to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

In another preferred embodiment, the antibody specific for CDK1 is a monoclonal or polyclonal antibody.

In another preferred embodiment, said CDK1 protein further comprises a derivative of CDK1 protein.

In another preferred embodiment, the derivative of CDK1 protein comprises a modified CDK1 protein, a protein molecule having an amino acid sequence homologous to a native CDK1 protein and having the activity of a native CDK1 protein, a fusion protein comprising the amino acid sequence of a CDK1 protein.

In another preferred embodiment, the modified CDK1 protein is a pegylated CDK1 protein.

In another preferred embodiment, the expression "a protein molecule having an amino acid sequence homologous to a native CDK1 protein and having the activity of a native CDK1 protein" means that the amino acid sequence has 85% or more homology, preferably 90% or more homology, more preferably 95% or more homology, most preferably 98% or more homology to the CDK1 protein; and protein molecules having the activity of a native CDK1 protein.

In another preferred embodiment, the diagnostic reagent or kit is further used for distinguishing an early stage gastrointestinal stromal tumor from a late stage gastrointestinal stromal tumor.

In a second aspect, the invention provides a diagnostic kit for detecting advanced gastrointestinal stromal tumors, comprising a container containing a detection reagent for detecting CDK1 gene, mRNA, cDNA, or protein; and a label or instructions indicating that the kit is for detecting advanced gastrointestinal stromal tumors.

In another preferred embodiment, the detection of advanced gastrointestinal stromal tumors refers to determining the likelihood of developing advanced gastrointestinal stromal tumors.

In another preferred example, the judgment includes a preliminary judgment (prediction).

In another preferred embodiment, the detection reagent for detecting CDK1 gene, mRNA, cDNA, or protein comprises:

(a) an antibody specific for CDK1 protein; and/or

(b) A specific primer that specifically amplifies mRNA or cDNA of CDK 1.

In another preferred embodiment, the test is a tissue sample test.

In another preferred embodiment, the CDK1 gene, mRNA, cDNA, or protein or its detection reagents can be used as a control or reference.

In another preferred embodiment, the diagnostic kit is further used for distinguishing between an early stage gastrointestinal stromal tumor and a late stage gastrointestinal stromal tumor.

In another preferred embodiment, the label or instructions indicates that the kit is to be used for:

(a) detecting advanced gastrointestinal stromal tumors;

(b) differentiate between early stage gastrointestinal stromal tumors and late stage gastrointestinal stromal tumors.

In another preferred embodiment, the test subject is a human or non-human mammal.

In a third aspect, the present invention provides a method of detecting advanced gastrointestinal stromal tumors, the method comprising:

a) providing a test sample from a subject;

b) detecting the expression level of CDK1 protein in the test sample E1; and

c) comparing the expression level of CDK1 protein determined in step b) with a reference value,

wherein expression of the CDK1 protein in the sample at an amount greater than the reference value indicates that the subject has advanced gastrointestinal stromal tumor.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the test sample is a cell or tissue of an advanced gastrointestinal stromal tumor.

In another preferred example, the reference value is a cut-off value (cut-off value).

In another preferred embodiment, the reference value is the relative expression level of CDK1 in the sample.

In another preferred embodiment, the reference value is 5(RNA level).

In another preferred example, the expression level of CDK1 RNA in the sample is detected by RT-PCR or transcriptome sequencing, and the expression level of CDK1 protein in the sample is detected by immunoblotting or immunohistochemistry.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

A fourth aspect of the invention provides a method of determining a treatment regimen comprising:

a) providing a test sample from a subject;

b) detecting the expression level of CDK1 protein in the test sample; and

c) determining a treatment regimen based on the expression level of CDK1 protein in the sample.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred example, where expression of the CDK1 protein in the sample is above a reference value, indicating that the subject has advanced gastrointestinal stromal tumors, the treatment regimen comprises CDK1 inhibitor therapy, a CDK1 inhibitor in combination with a tyrosine kinase inhibitor therapy.

In another preferred embodiment, the CDK1 inhibitor therapy, CDK1 inhibitor therapy in combination with a tyrosine kinase inhibitor is selected from the group consisting of:

CDK1 inhibitor therapy: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof;

tyrosine kinase inhibitor therapy: a small molecule compound selected from the group consisting of: imatinib, sunitinib, regorafenib, Avapritinib (BLU-285), Ripretinib (DCC-2618), or combinations thereof.

In another preferred example, when the subject has advanced gastrointestinal stromal tumors, the treatment regimen further comprises CDK1 inhibitor therapy, a CDK1 inhibitor in combination with a tyrosine kinase inhibitor therapy; and other medicines for treating advanced gastrointestinal stromal tumor.

In another preferred embodiment, the other drug for treating advanced gastrointestinal stromal tumors is selected from the group consisting of: imatinib, sunitinib, regorafenib, atorvastatin (Avapritinib, BLU-285), ripatinib (Ripretinib, DCC-2618), or combinations thereof.

In a fifth aspect, the invention provides the use of an inhibitor of the CDK1 gene or protein thereof in the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is selected from the group consisting of: antibodies, small molecule compounds, microRNAs, siRNAs, shRNAs, or combinations thereof.

In another preferred embodiment, the inhibitor comprises an inhibitor that inhibits the expression of CDK1 gene or a protein thereof.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is selected from the group consisting of: RO-3306,

NU6027, Flavopiridol, AT7519, GW5074, Dinaciclib, JNJ-7706621, AZD5438, BMS-265246, R547, CDKI-73, Alsterpaullone, SU9516, or a combination thereof.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the composition comprises a therapeutically effective amount of an inhibitor of the CDK1 gene or protein thereof, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the medicament is administered by a mode of administration selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, topical or systemic transdermal administration of the skin.

In another preferred embodiment, the formulation is selected from the group consisting of: tablet, capsule, injection, granule, and spray.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising:

(a1) an inhibitor of the CDK1 gene or protein thereof;

(a2) tyrosine kinase inhibitors; and

(b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises:

(c) other drugs for preventing and/or treating advanced gastrointestinal stromal tumors.

In another preferred embodiment, the tyrosine kinase inhibitor is selected from the group consisting of: imatinib, sunitinib, regorafenib, Avapritinib (BLU-285), Ripretinib (DCC-2618), or combinations thereof.

In another preferred embodiment, the weight ratio of component (a1) to component (a2) is 100:1 to 0.01:1, preferably 10:1 to 0.1:1, more preferably 2:1 to 0.5: 1.

In another preferred embodiment, the content of the component (a1) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of the component (a2) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of the component (c) in the pharmaceutical composition is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, in the pharmaceutical composition, the component (a1) and the optional component (a2) and the optional component (c) account for 0.01 to 99.99 wt%, preferably 0.1 to 90 wt%, more preferably 1 to 80 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the dosage forms of the pharmaceutical composition include injection dosage forms and oral dosage forms.

In another preferred embodiment, the oral dosage form comprises tablets, capsules, films, and granules.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a sustained release dosage form and a non-sustained release dosage form.

A seventh aspect of the invention provides a product combination comprising:

(i) a first pharmaceutical composition comprising (a) a first active ingredient which is an inhibitor of the CDK1 gene or protein thereof, and a pharmaceutically acceptable carrier; and

(ii) a second pharmaceutical composition comprising (b) a second active ingredient which is a tyrosine kinase inhibitor, and a pharmaceutically acceptable carrier;

wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions or the same pharmaceutical composition.

In another preferred embodiment, the weight ratio of the component (i) to the component (ii) is 100:1 to 0.01:1, preferably 10:1 to 0.1:1, more preferably 2:1 to 0.5: 1.

In another preferred embodiment, the content of the component (i) in the product combination is 1% to 99%, preferably 10% to 90%, more preferably 30% to 70%.

In another preferred embodiment, the content of the component (ii) in the product combination is 1% to 99%, preferably 10% to 90%, and more preferably 30% to 70%.

In another preferred embodiment, the component (i) and the component (ii) in the product combination represent 0.01 to 99.99 wt%, preferably 0.1 to 90 wt%, more preferably 1 to 80 wt% of the total weight of the product combination.

In another preferred embodiment, the product combination further comprises a detection reagent of CDK1 or a kit thereof.

In another preferred embodiment, the kit comprises a container comprising detection reagents for detecting the CDK1 gene, mRNA, cDNA, or protein; and a label or instructions indicating that the kit is for detecting advanced gastrointestinal stromal tumors.

An eighth aspect of the present invention provides a medicine cartridge comprising:

(a1) a first container, and an inhibitor of the CDK1 gene or protein thereof, or a medicament comprising an inhibitor of the CDK1 gene or protein thereof, located in said first container;

(b1) a second container, and a tyrosine kinase inhibitor, or a medicament containing a tyrosine kinase inhibitor, located in the second container.

In another preferred embodiment, the kit further comprises:

(c1) a third container, and other medicines for preventing and/or treating advanced gastrointestinal stromal tumors or medicines containing other medicines for preventing and/or treating advanced gastrointestinal stromal tumors in the third container.

In another preferred embodiment, the kit further comprises (d1) a fourth container, and a detection reagent for CDK1 located in said fourth container.

In another preferred embodiment, the first container and the second, third and fourth containers are the same or different containers.

In another preferred embodiment, the drug of the first container is a single formulation comprising an inhibitor of the CDK1 gene or protein thereof.

In another preferred embodiment, the drug in the second container is a single formulation containing a tyrosine kinase inhibitor.

In another preferred embodiment, the drug in the third container is a single preparation containing other drugs for preventing and/or treating advanced gastrointestinal stromal tumor.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit further comprises instructions.

In another preferred embodiment, the description recites one or more descriptions selected from the group consisting of:

(a) use of an inhibitor of the CDK1 gene or protein thereof to (i) inhibit the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) methods of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(b) combining an inhibitor of the CDK1 gene or protein thereof with a tyrosine kinase inhibitor, and/or optionally other prophylactic and/or therapeutic agent for the advanced gastrointestinal stromal tumor (i) to inhibit growth or proliferation of cells of the advanced gastrointestinal stromal tumor; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(c) detecting expression levels of a CDK1 protein in a patient having advanced gastrointestinal stromal tumor while administering an inhibitor of the CDK1 gene or protein thereof to (i) inhibit growth or proliferation of cells of the advanced gastrointestinal stromal tumor; and/or (ii) the prevention and/or treatment of advanced gastrointestinal stromal tumors; and/or (iii) methods of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors;

(d) detecting the expression level of a CDK1 protein in a patient with advanced gastrointestinal stromal tumor in combination with an inhibitor of the CDK1 gene or protein thereof; and a tyrosine kinase inhibitor, and/or optionally other prophylactic and/or therapeutic agent for advanced gastrointestinal stromal tumor to (i) inhibit growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) methods of increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

A ninth aspect of the invention provides a pharmaceutical composition according to the sixth aspect of the invention or a product combination according to the seventh aspect of the invention or a kit according to the eighth aspect of the invention for use in the preparation of a medicament for (i) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) agents that increase the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

In another preferred embodiment, the inhibitor of the CDK1 gene or its protein in the pharmaceutical composition has an effect concentration of 3000ng/ml, preferably 200 ng/ml, more preferably 350 ng/ml and 500 ng/ml.

In another preferred embodiment, the concentration of the tyrosine kinase inhibitor in the pharmaceutical composition is 10-100000ng/ml, preferably 100-10000ng/ml, more preferably 500-2000 ng/ml.

In another preferred embodiment, the effective concentration of the other drugs for preventing and/or treating advanced gastrointestinal stromal tumor in the pharmaceutical composition is 10-100000ng/ml, preferably 100-10000ng/ml, more preferably 500-2000 ng/ml.

In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of the CDK1 gene or protein thereof; and (b) optionally a tyrosine kinase inhibitor; and (c) optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors; and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, in said pharmaceutical composition or kit, said inhibitor of CDK1 gene or protein thereof; and (b) optionally a tyrosine kinase inhibitor; and (c) optionally other drugs for preventing and/or treating advanced gastrointestinal stromal tumors, in an amount of 0.01-99.99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% based on the total weight of the pharmaceutical composition or kit.

In a tenth aspect, the present invention provides a method for preventing and/or treating advanced gastrointestinal stromal tumors, comprising:

administering to a subject in need thereof an inhibitor of the CDK1 gene or protein thereof; or a pharmaceutical composition according to the sixth aspect of the invention or a product combination according to the seventh aspect of the invention or a kit according to the eighth aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal having advanced gastrointestinal stromal tumors.

In another preferred embodiment, the non-human mammal includes rodents and primates, preferably mice, rats, rabbits, monkeys.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered in a dose of 0.5-10 mg/kg body weight, preferably 1-6mg/kg body weight, most preferably 3-5mg/kg body weight.

In another preferred embodiment, the tyrosine kinase inhibitor is administered in a dose of 1-600mg/kg body weight, preferably 2-60mg/kg body weight, most preferably 3-10mg/kg body weight.

In another preferred embodiment, the other agent for preventing and/or treating advanced gastrointestinal stromal tumors is administered in a dose of 0.06-600mg/kg body weight, preferably 1-60mg/kg body weight, most preferably 3-12mg/kg body weight.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered at a frequency of 2-5 times per week, preferably 3-4 times per week.

In another preferred embodiment, the tyrosine kinase inhibitor is administered at a frequency of 5-20 times per week, preferably 10-15 times per week.

In another preferred embodiment, the other medicament for preventing and/or treating advanced gastrointestinal stromal tumors is administered at a frequency of 3-15 times/week, preferably 6-10 times/week.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered for a period of 20 to 90 days, preferably 20 to 60 days, most preferably 30 to 40 days.

In another preferred embodiment, the tyrosine kinase inhibitor is administered for a period of 20 to 90 days, preferably 20 to 60 days, and most preferably 30 to 40 days.

In another preferred embodiment, the other agent for preventing and/or treating advanced gastrointestinal stromal tumors is administered for 20 to 90 days, preferably 20 to 60 days, and most preferably 30 to 40 days.

In another preferred embodiment, the inhibitor of the CDK1 gene or protein thereof is administered simultaneously or sequentially with an optional tyrosine kinase inhibitor, and optionally other agents for the prevention and/or treatment of advanced gastrointestinal stromal tumors.

In an eleventh aspect, the invention provides an in vitro non-therapeutic method of inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells, comprising the steps of: culturing advanced gastrointestinal stromal tumor cells in the presence of a CDK1 gene or a protein inhibitor thereof, thereby inhibiting growth or proliferation of gastrointestinal stromal tumor cells.

In another preferred embodiment, said CDK1 gene or protein inhibitor thereof is selected from the group consisting of: antibodies, small molecule compounds, microRNAs, siRNAs, shRNAs, or combinations thereof.

In another preferred embodiment, the advanced gastrointestinal stromal tumor cells highly express CDK1 protein.

In another preferred embodiment, the method further comprises adding a tyrosine kinase inhibitor to the culture system of the advanced gastrointestinal stromal tumor cells; and/or other agents that prevent and/or treat advanced gastrointestinal stromal tumors, thereby inhibiting the growth or proliferation of cells of the advanced gastrointestinal stromal tumors.

In another preferred example, the advanced gastrointestinal stromal tumor cells are cells cultured in vitro.

In a twelfth aspect of the present invention, there is provided a method of screening for a candidate compound for the prevention and/or treatment of advanced gastrointestinal stromal tumors, the method comprising the steps of:

(a) in the test group, a test compound is added to a culture system of cells, and the expression amount (E1) and/or activity (a1) of CDK1 is observed in the cells of the test group; in the control group, no test compound was added to the culture system of the same cells, and the expression amount (E0) and/or activity (a0) of CDK1 in the cells of the control group were observed;

wherein, if the expression level (E1) and/or activity (A1) of CDK1 of the cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and/or treating advanced gastrointestinal stromal tumors having an inhibitory effect on the expression and/or activity of CDK 1.

In another preferred embodiment, the expression level of CDK1 is determined by quantitative fluorescence PCR or immunohistochemistry or immunoblot detection.

In another preferred example, the method further comprises the steps of:

(b) further testing the candidate compound obtained in step (a) for inhibition of growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or further tested for its effect on CDK1 gene downregulation.

In another preferred example, step (b) includes the steps of: in the test group, adding a test compound into a culture system of the advanced gastrointestinal stromal tumor cells, and observing the number and/or growth condition of the advanced gastrointestinal stromal tumor cells; in the control group, no test compound was added to the culture system of the advanced gastrointestinal stromal tumor cells, and the number and/or growth of the advanced gastrointestinal stromal tumor cells were observed; wherein, if the number or growth rate of the advanced gastrointestinal stromal tumor cells in the test group is less than that in the control group, the test compound is a candidate compound for preventing and/or treating the advanced gastrointestinal stromal tumor having an inhibitory effect on the growth or proliferation of the advanced gastrointestinal stromal tumor cells.

In another preferred embodiment, the method comprises the step (c): administering the candidate compound identified in step (a) to a mammalian model and determining its effect on the mammal.

In another preferred embodiment, the mammal is a mammal with advanced gastrointestinal stromal tumors.

In another preferred embodiment, the phrase "substantially less than" means E1/E0 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.

In another preferred embodiment, the phrase "significantly less than" means A1/A0. ltoreq. 1/2, preferably. ltoreq. 1/3, more preferably. ltoreq. 1/4.

In another preferred embodiment, the cells comprise advanced gastrointestinal stromal tumor cells.

In another preferred embodiment, the cells are cultured in vitro.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows (A) a volcano plot of RNA-seq showing the pattern of differential gene expression between early and late GISTs. (B) Pattern diagram of whole genome CRISPR screening of advanced GIST cells. (C) Boxplots show the distribution of sgrnas at different doublings. (D) The CRISPR score for each gene was ranked in the genome-wide CRISPR screen of GIST430/654 cells. CDK1 ranks second. All genes analyzed in the CRISPR screen are identified in grey, 568 genes identified in the entire transcriptome sequencing that are highly expressed in late GIST are identified in black.

Figure 2 shows the expression of CDK1, CDK4, CDK6 and CDK9 in the GIST discovery cohort (early GIST n 11, late n 32) with CDK1 being significantly more highly expressed in late GIST than in early GIST compared to other CDKs. (B) Once CDK1 was highly expressed in one metastasis, it was highly expressed in all other metastases of the same GIST patient. (C) Ki67 is a molecular marker of proliferation and CDK1 and Ki67 expression are highly correlated (D) western blots (n ═ 92) showing that CDK1 is not/poorly expressed in early GIST, but is frequently expressed in late GIST. (E) Immunohistochemical examination of formalin fixed paraffin embedded GIST clinical samples revealed high expression of CDK1 in late GIST, whereas CDK1 expression levels were very low in early GIST. (F) Statistical analysis of tissue chip samples containing 325 early GIST and 177 late GIST. High expression of CDK1 was highly correlated with the malignancy of GIST.

FIG. 3 shows (A) the detection of the knockdown of CDK1 in imatinib-resistant GIST430/654 and imatinib-sensitive GIST-T1 cell lines by qRT-PCR. (B) Lentivirally mediated CDK1 knockdown significantly reduced the viability of GIST430/654 and GIST-T1 cells in a short period of time, as assessed by the CellTiter-Glo viability assay. (C) Crystal violet staining showed CDK1 knockdown long-term inhibition of GIST cell proliferation. (D) CDK1 knockdown inhibited anchorage-independent growth of GIST430/654 and GIST-T1 cells. (E) CDK1 knockdown inhibited tumor growth in GIST430/654 and GIST-T1 xenografted mice. (F) Cell cycle analysis showed that CDK1 knock-down decreased the cell proportion of GIST430/654 and GIST-T1 in S phase and G2/M phase, and increased the cell proportion of G0/G1 phase, resulting in G0/G1 blockade. (G) Cytosenescence staining analysis showed that CDK1 knockdown significantly increased the proportion of senescent cells of GIST430/654 and GIST-T1, promoting cellular senescence, which is also one of the causes of G0/G1 cell cycle arrest.

Figure 4 shows (a) identification of proteins interacting with CDK1 by co-immunoprecipitation and mass spectrometry-based proteomics. GIST430/654 cells were lentivirally transduced with FLAG-CDK1 and subjected to a pull-down assay using FLAG antibody. The table shows the proteins with high confidence in the mass spectra, AKT1 ranked second. (B) validation of AKT-CDK1 interaction in PDK1 normal and PDK1 deficient GIST430/654 cells. GIST430/654 cells were transduced with FLAG-CDK1 and cell lysates were co-immunoprecipitated using FLAG antibody, followed by immunoblotting using AKT antibody, indicating that the binding of CDK1 and AKT was independent of PDK 1. (C) Treatment with MK-2206, an inhibitor of AKT, inhibits the binding of CDK1 to AKT and reduces the level of phosphorylation of AKT in PDK1 normal GIST430/654 cells. (D) Endogenous CDK1 strongly interacted with AKT in GIST430/654 cell line. Cells treated with control or CDK1 inhibitor (RO-3306, 1 μ M) were immunoblotted with CDK1 and AKT IP. Left, cell lysates were immunoprecipitated with CDK1 antibody and immunoblotted with AKT antibody. On the right, cell lysates were immunoprecipitated with AKT antibody and immunoblotted with CDK1 antibody. AKT1 was shown to interact with CDK1 in the GIST system. (E) In vitro interaction assays also showed that CDK1 interacts with AKT and that this interaction is attenuated upon RO-3306 inhibition. . (F) Co-immunoprecipitation in 293 cells following exogenous introduction of FLAG-CDK1 and HA-AKT1 showed strong interaction between CDK1 and AKT1, and CDK1 promoted phosphorylation of threonine 308 and serine 473 of AKT. (G) In PDK1 knock-out GIST430/654 cells, co-immunoprecipitation showed strong interaction between CDK1 and AKT1 and promoted phosphorylation of threonine 308 and serine 473 of AKT following exogenous introduction of FLAG-CDK1 and HA-AKT 1. Indicating that CDK1 can activate AKT alone independent of PDK 1. (H) Knock-down of CDK1 in GIST430/654 and GIST-T1 cells inhibited AKT phosphorylation, induced apoptosis, and reduced PCNA expression. (I) Treatment of GIST430/654 and GIST-T1 cells with RO-3306 inhibited AKT phosphorylation, induced apoptosis, and inhibited cell proliferation as did CDK1 knockdown.

FIG. 5 shows (A) complementation of AKT following CDK1 knock-down in GIST430/654, complementing the phenotype of inhibited cell viability following CDK1 knock-down. The viability of GIST430/654 cells was assessed by the CellTiter-Glo viability assay. (B) After CDK1 was knocked down in GIST430/654, AKT was complemented, complementing the phenotype of inhibition of cell proliferation after CDK1 knock-down. GIST430/654 cell proliferation was assessed by a crystal violet staining assay. (C) After CDK1 was knocked down in GIST430/654, AKT was recruited to compensate for the phenotype independent of inhibition of cell anchorage after CDK1 knock down. GIST430/654 cell anchorage independent growth was assessed by soft agar experiments. (D) After CDK1 is knocked down in GIST430/654, AKT is supplemented, and the tumor growth of xenograft mice is inhibited after CDK1 is knocked down. CDK1 was shown to promote growth and proliferation of GIST cells by AKT.

FIG. 6 shows (A) the CellTiter-Glo growth inhibition assay for the CDK1 inhibitor RO-3306 in GIST cell lines and GIST primary cells. GIST-1, BJ was established from metastatic GISTs without CDK1 expression as a non-transformed fibroblast cell line. RO-3306 inhibited imatinib-sensitive and resistant GIST cell lines, without affecting GIST primary cells and fibroblasts that do not express CDK 1. (B) Half maximal inhibitory concentration of RO-3306 against imatinib-resistant and imatinib-sensitive GIST cells (IC 50). (C) Immunoblots showed expression levels of CDK1 in GIST cells as well as GIST primary cells. (D) Immunoblotting showed that RO-3306 treated imatinib-resistant and imatinib-sensitive GIST cells did not affect AKT expression, but significantly reduced the level of AKT phosphorylation. (E-G) antitumor activity of CDK1 inhibitors on imatinib-resistant GIST xenograft mice. Treatment of imatinib-resistant patient-derived xenograft GIST tumor-bearing mice with CDK1 inhibitors, patient-derived xenograft GIST tumor-bearing mice (ex 11+ ex 17) were treated with 4mg/kg RO-3306 every 2 days or 50mg/kg imatinib twice daily, reducing tumor volume and tumor size. And CDK1 inhibitors did not affect the body weight changes of imatinib-resistant GIST xenograft mice. (H) Combined treatment of GIST-T1 cells with RO-3306 and imatinib significantly inhibited GIST cell growth compared to treatment alone. (I-K) antitumor Activity of RO-3306 against Imatinib-sensitive GIST xenograft mice. Mice bearing GIST-T1 tumors were orally administered twice daily as single drug or combination therapy with 4mg/kg RO-3306 or a low dose (25mg/kg) of imatinib every 2 days. Treatment of imatinib-sensitive GIST-T1 tumor-bearing mice with a CDK1 inhibitor in combination with imatinib reduced tumor volume as well as tumor size. And CDK1 inhibitors did not affect changes in body weight in imatinib-sensitive GIST xenograft mice. The CDK1 inhibitor was shown to have anti-tumor activity against imatinib-resistant and imatinib-sensitive advanced GIST.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that the expression of the gene of CDK1 or its protein in late gastrointestinal stromal tumor cells or tissues is significantly higher than the expression of the gene of CDK1 or its protein in early gastrointestinal stromal tumor tissues, and thus, the CDK1 gene or its protein can be used as a marker for detecting late gastrointestinal stromal tumors. Moreover, applicants have also surprisingly found that inhibitors of the CDK1 gene or protein thereof are effective in (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increasing the resistance and sensitivity of cells of advanced gastrointestinal stromal tumors to tyrosine kinase inhibitors, and the inhibitor of the CDK1 gene or protein thereof may be used in combination with a tyrosine kinase inhibitor and/or optionally other agents for preventing and/or treating advanced gastrointestinal stromal tumors, and has a significant synergistic effect in the treatment of advanced gastrointestinal stromal tumors. On this basis, the present inventors have completed the present invention.

As used herein, the term "RO-3306" has the formula

NU6027 has the structural formula

The structural formula of Flavopiridol is shown in the specification

AT7519 has a structural formula of

The structural formula of GW5074 is

The structural formula of the Dinaciclib is shown as

The structural formula of JNJ-7706621 is

The structural formula of AZD5438 is

BMS-265246 has a structural formula

R547 has the structural formula

The structural formula of CDKI-73 is

The structural formula of Alsterpaullone is shown as

SU9516 has a structural formula

The structural formula of Avapritinib (BLU-285) is shown in the specification

DCC-2618 has the structural formula

Gastrointestinal stromal tumor

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the stomach and intestine, clinically symptomatic gastrointestinal stromal tumors occur in adults over 45 years old, gastrointestinal stromal tumors can occur in the whole digestive tract, but most of the gastrointestinal stromal tumors occur in the stomach and small intestine, clinical stromal tumors are the most common sarcomas, and the annual incidence rate is 10-20/100 ten thousands of people. With the progress of endoscopic technology and imaging technology, more and more gastrointestinal stromal tumors with smaller volumes are discovered. Multiple pathological studies have proved that small gastrointestinal stromal tumors with a diameter less than 1cm are common in middle-aged and elderly people, the discovery rate can reach 35%, and with the aging of the world population, 1 hundred million patients with the small gastrointestinal stromal tumors in China are estimated.

The main pathogenesis of gastrointestinal stromal tumor is activation mutation of protooncogene KIT in Kahar stromal cells, so that downstream signal pathways are abnormally activated, mainly comprising a MAPK pathway and a PI3K-AKT pathway, and the survival, growth and proliferation of cells are out of control.

Advanced gastrointestinal stromal tumor

The gastrointestinal stromal tumors are divided into primary gastrointestinal stromal tumors and metastatic gastrointestinal stromal tumors according to the existence of metastasis, the primary gastrointestinal stromal tumors are divided into low-risk, medium-risk and high-risk gastrointestinal stromal tumors according to pathological indexes (tumor size, mitosis number per high-power visual field, anatomical position and the like), and the high-risk gastrointestinal stromal tumors have high risk level of metastasis and recurrence. The high-risk gastrointestinal stromal tumor and the metastatic gastrointestinal stromal tumor are collectively called advanced gastrointestinal stromal tumor. The prognosis of the advanced gastrointestinal stromal tumor is poor, and a treatment means is urgently needed in clinic. About 80% of gastrointestinal stromal tumors contain KIT activating mutations, molecular targeted therapy targeting KIT oncoproteins revolutionized the treatment of advanced stromal tumors, but the molecular heterogeneity among gastrointestinal stromal tumor individuals led to highly inconsistent response of different individuals to targeted therapy. The gene detection plays an important role in predicting the curative effect of the gastrointestinal stromal tumor targeted therapy, disease prognosis and the like. At present, almost all patients suffering from gastrointestinal stromal tumor receiving targeted therapy develop drug resistance, and how to improve the curative effect of targeted therapeutic drugs represented by imatinib is an urgent problem to be solved in clinical medicine and basic medicine.

Sample(s)

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner such that the test performed on the sample provides information about the subject. The sample may also be a material that has been contacted with other materials that are not the subject, but that enable the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a probe or scalpel wash. The sample can be a source of biological material other than that contacted with the subject, so long as one skilled in the art is still able to determine information about the subject from the sample.

Expression of

As used herein, the term "expression" includes the production of mRNA from a gene or portion of a gene, and includes the production of protein encoded by an RNA or gene or portion of a gene, as well as the presence of a test substance associated with expression. For example, cDNA, binding of a binding partner (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic moieties of the binding partner are included within the scope of the term "expression". Thus, an increase in the density of half-spots on immunoblots such as western blots is also within the scope of the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically related to a particular result when compared to the results of an analysis. In preferred embodiments, the reference value is determined from a statistical analysis performed on studies comparing the expression of CDK1 protein to known clinical outcomes. Some of these studies are shown in the examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust the reference values. Reference values can also be determined by considering conditions and outcomes particularly relevant to the patient's medical history, genetics, age, and other factors.

In the present invention, the reference value refers to a cut-off value, which refers to the relative expression level of CDK1 in cells or tissues of advanced gastrointestinal stromal tumors, preferably the relative expression level is 5 (FPKM value obtained by transcriptome sequencing, FPKM: Fragments Per Kilobase of exon model Per Million mapped Fragments, i.e., Fragments read Per Million maps Per Kilobase of transcription, reflecting RNA expression level at a certain level)

Samples of non-advanced gastrointestinal stromal tumors

As used herein, the term "non-advanced gastrointestinal stromal tumor sample" includes, but is not limited to, a population not having advanced gastrointestinal stromal tumors, non-advanced gastrointestinal stromal tumor tissue from patients with advanced gastrointestinal stromal tumors.

CDK1 proteins and polynucleotides

In the present invention, the terms "protein of the invention", "CDK 1 protein", "CDK 1 polypeptide" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence CDK 1. They include CDK1 proteins with or without the initial methionine. In addition, the term also includes full-length CDK1 and fragments thereof. The CDK1 protein of the present invention includes its complete amino acid sequence, its secreted protein, its mutants and its functionally active fragments.

CDK1 is a cyclin-dependent kinase 1 that functions as a serine/threonine kinase and is a key player in cell cycle regulation. In humans, CDK1 is encoded by the CDC2 gene. It is essential for the G1/S and G2/M phase transitions of the eukaryotic cell cycle. CDK1 forms a complex with cyclins that phosphorylates a variety of target substrates (more than 75 have been identified in germinating yeast). Phosphorylation and dephosphorylation of this protein plays an important regulatory role in cell cycle control.

The full length of the human CDK1 protein was 297 amino acids (accession No. NP _ 001777). The full length of the murine CDK1 protein was 297 amino acids (accession No. NP _ 031685).

In the present invention, the terms "CDK 1 gene", "CDK 1 polynucleotide" are used interchangeably and all refer to a nucleic acid sequence having the nucleotide sequence of CDK 1.

The genome of the human CDK1 gene has a total length of 23522bp (NCBI GenBank accession No. NG _029877.1), and the mRNA sequence of the transcription product has a total length of 1889bp (NCBI GenBank accession No. NM _ 001786).

The genome total length of the mouse CDK1 gene is 17767bp (NCBI GenBank accession number NC-000076.7), and the transcription product mRNA sequence total length is 4362bp (NCBI GenBank accession number NM-007659.4).

Human and murine CDK1 showed 76.25% similarity at the DNA level and 96.97% protein sequence similarity.

It is understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. It is also understood that nucleotide changes may be acceptable when conservative amino acid substitutions are made by nucleotide substitutions.

When an amino acid fragment of CDK1 is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the CDK1 nucleotide sequence disclosed herein, particularly the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments, derivatives thereof) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the present invention may be used to express or produce recombinant CDK1 polypeptides by conventional recombinant DNA techniques. Generally, the following steps are provided:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human CDK1 polypeptide, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the CDK1 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the CDK1 encoding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; an insect cell; animal cells, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods of treatment, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, it is possible to useThe following DNA transfection methods were selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Specific antibodies

In the present invention, the terms "antibody of the invention" and "antibody specific against CDK 1" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for a human CDK1 polypeptide. Herein, "specificity" means that the antibody binds to the human CDK1 gene product or fragment. Preferably, these antibodies bind to the human CDK1 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules capable of binding to and inhibiting human CDK1 protein, as well as those antibodies that do not affect the function of human CDK1 protein. The invention also includes those antibodies which bind to the human CDK1 gene product in modified or unmodified form.

The present invention encompasses not only intact monoclonal or polyclonal antibodies,but also antibody fragments with immunological activity, such as Fab' or (Fab) ₂ A fragment; an antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but retain portions of the antibody from a human.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified human CDK1 gene product, or antigenic fragment thereof, may be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing the human CDK1 protein or antigenic fragment thereof may be used to immunize animals to produce antibodies. The antibody of the present invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al,Nature256 of; 495, 1975; in the case of the plant of Kohler et al,Eur.J.Immunol.6: 511,1976; in the case of the plant of Kohler et al,Eur.J.Immunol.6: 292, 1976; in Hammerling et al,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). Antibodies of the invention include antibodies that block the function of human CDK1 protein as well as antibodies that do not affect the function of human CDK1 protein. The antibodies of the invention may be obtained by conventional immunological techniques using fragments or functional regions of the human CDK1 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human CDK1 gene product may be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Antibodies against human CDK1 protein may be used in immunohistochemical techniques to detect human CDK1 protein in a sample, particularly a tissue sample or a serum sample.

Detection method

The invention also provides a method for detecting advanced gastrointestinal stromal tumors by utilizing the characteristic that CDK1 is highly expressed in cells or tissues of the advanced gastrointestinal stromal tumors.

In a preferred embodiment of the invention, the invention provides a high-throughput next generation sequencing method for detecting CDK1, Sanger sequencing, quantitative fluorescence pcr (qpcr), immunoblotting, in situ immunofluorescence (FISH), immunohistochemistry, and the like.

Detection kit

Based on the relevance of CDK1 to advanced gastrointestinal stromal tumors, CDK1 is present in advanced gastrointestinal stromal tumor tissues, CDK1 may be a diagnostic marker for advanced gastrointestinal stromal tumors.

The invention also provides a kit for detecting the advanced gastrointestinal stromal tumor, which contains a detection reagent for detecting CDK1 gene, mRNA, cDNA or protein; and a label or instructions indicating that the kit is for detecting advanced gastrointestinal stromal tumors.

Wherein the label or the instruction indicates that the kit is used for detecting advanced gastrointestinal stromal tumors.

Detection method and kit

The present invention relates to diagnostic assays for quantitative and in situ measurement of human CDK1 protein levels or mRNA levels. These assays are well known in the art. Human CDK1 protein levels detected in the assay may be used to diagnose (including aiding diagnosis) advanced gastrointestinal stromal tumors.

One method of detecting the presence or absence of a CDK1 protein in a sample is by detection using antibodies specific for the CDK1 protein, which comprises: contacting the sample with an antibody specific for a CDK1 protein; observing whether an antibody complex is formed indicates the presence of CDK1 protein in the sample.

The CDK1 protein or polynucleotide thereof can be used for diagnosing and treating diseases related to CDK1 protein. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and gene diagnosis. Antibodies against CDK1 may be immobilized on a protein chip for detecting CDK1 protein in a sample.

The main advantages of the present invention include:

(1) the invention discovers for the first time that the expression of the gene of CDK1 or the protein thereof in advanced gastrointestinal stromal tumor cells or tissues is significantly higher than that of the gene of CDK1 or the protein thereof in normal tissues, so the CDK1 gene or the protein thereof can be used as a marker for detecting advanced gastrointestinal stromal tumors.

(2) The invention has found for the first time that an inhibitor of the CDK1 gene or protein thereof is effective in (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

(3) The invention discovers for the first time that an inhibitor of CDK1 gene or protein thereof can be combined with a tyrosine kinase inhibitor and/or other optional medicines for preventing and/or treating advanced gastrointestinal stromal tumors, and has a remarkable synergistic effect on the treatment of the advanced gastrointestinal stromal tumors.

(4) The invention discovers for the first time that CDK1 is highly expressed in a tissue sample of a patient with advanced gastrointestinal stromal tumor.

(5) The CDK1 is found to promote the growth and proliferation of gastrointestinal stromal tumor cells for the first time.

(6) The invention discovers for the first time that inhibiting the expression or activity of CDK1 inhibits the growth and proliferation of advanced gastrointestinal stromal tumors (GIST).

(7) The invention discovers for the first time that inhibition of the expression or activity of CDK1 can significantly improve the resistance and sensitivity of GIST to tyrosine kinase inhibitors (such as imatinib).

(8) The invention finds that CDK1 promotes GIST growth and proliferation through AKT for the first time.

(9) The present invention has for the first time found that CDK1 binds and modulates AKT phosphorylation.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.

Example 1 genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screening, CDK1 was found to be a target for advanced gastrointestinal stromal tumors (GIST)

To find functional genes affecting GIST cells, this study used CRISPR/Cas9 for inactivation screening of GIST430/654 cells. GIST430/654 cells were infected with GeCKOv2 library virus at an MOI of about 0.4, and all cells were divided into 2 groups after puromycin (puromycin) screening, with the number of cells per group not less than 300 Xthe coverage of the library. The first group extracted cell genomic DNA as Day 0 control, and the second group extracted cell genomic DNA after 15 serial culture. The genome DNA is subjected to nested PCR to amplify the introduced sgRNA, and a barcode sequence is introduced, and the amplicon is subjected to next generation sequencing with the depth of more than 300 times. Based on changes in sgRNA reads during CRISPR/Cas9 inactivation screening, CRISPR gene score (CS) was calculated and used to reflect gene importance.

The experimental steps are as follows:

1) transcriptome data was analyzed for 43 GIST samples (fig. 2A-C);

2) performing genome-wide CRISPR screening by using a late GIST cell line (GIST430/654) to knock out genes inhibiting cell proliferation;

3) the vulnerability of the late GIST was screened using a combination of transcriptome data and CRISPR screening data (FIGS. 1A-D).

The results of the experiment are shown in FIGS. 1A-D.

The results showed that 568 genes were highly expressed in late GIST and less expressed in early GIST (FIG. 1A); screening for advanced imatinib resistant GIST430/654 cells using the method of CRISPR screen of fig (1B); after 15 Population Doublings (PD), the distribution of sgrnas changed significantly compared to PD0 cells, indicating that the functional screen was consistent with the design (fig. 1C); in the GIST430/654 screening results, the CS value of CDK1 was ranked second (FIG. 1D). Overall CDK1 was shown to be a potential target for advanced gastrointestinal stromal tumors (GIST).

Example 2 significantly high expression of CDK1 in late GIST

Immunoblotting experiments: the harvested samples or cell proteins were added with IP buffer, lysed at 4 degrees overnight, centrifuged at 12000rpm for 30min, and Quick Start used ^TM Bradford 1 × Dye Reagent (Bio-Rad; #5000205) quantitated proteins. Electrophoresis and western blotting were performed using standard techniques. The hybridization signal was detected by chemiluminescence (Immobilon Western, Mi llipore Corporation, Mass.), and the luminescence signal was captured using an Amersham Imager 600 Imager (GE Healthcare; # 29083461).

Immunohistochemistry: tissues and tumor sections were immunohistochemically performed using CDK1 antibody (Santa Cruz # sc-54). Xylene was used for dewaxing and washing in a series of ethanol of different concentrations. Slides were boiled in citrate buffer (pH 6) by microwave for 12 minutes. Immunohistochemical reactions were visualized by diaminobenzidine staining.

The experimental steps are as follows:

1) CDK1 expression levels were detected by Western blotting in early and late GIST (fig. 2D);

2) CDK1 expression levels were detected by immunohistochemistry in early and late GIST (fig. 2E);

3) CDK1 expression levels were measured by 502 tissue chip samples of GIST (fig. 2F).

The results of the experiment are shown in FIGS. 2 (A-F).

The results showed that the 43 GIST sample RNA-seq results showed that only CDK1 was highly expressed (RNA levels) in late GIST relative to the other CDKs (fig. 2A); CDK1 was found to be highly expressed in one metastasis and CDK1 was also found to be highly expressed in the remaining metastases in one patient (fig. 2B); CDK1 expression was significantly positively correlated with KI67 expression (fig. 2C); immunoblot results of 92 samples showed that the proportion of expression levels in late GIST was 38/52 (72%), significantly higher than in early GIST (protein levels) (fig. 2D); 502 TMA immunohistochemistry chips showed that high expression of CDK1 was significantly associated with early and late GIST, with high expression in late GIST (fig. 2, E-F). All 3 cohorts showed significantly high expression of CDK1 in late GIST.

Example 3 deletion of the CDK1 Gene inhibits GIST growth and proliferation

And (3) slow virus packaging: transfection of 293T cells was mediated with Polyethyleneimine (PEI). Cells were plated at appropriate density on 10cm dishes one day before transfection, cells were changed with serum-free medium when they grew to about 70-90%, and plasmids were transfected with PEI 2 hours later. The lentiviral packaging plasmids were 9. mu.g.delta.8.9 and 3.5. mu.g vsv-g, 10. mu.g of the plasmid of interest. Complete culture medium is used for replacing the medium 4-6 hours after transfection, and virus solution is obtained by collecting supernatant after 24, 36, 48 and 60 hours.

And (3) detecting the cell viability: detection was performed using CellTiter-glo (CTG) kit. And (3) incubating the treated 96-well plate cells for 30 minutes at room temperature, diluting the CTG reagent by 4 times by using PBS, adding 100 mu L of diluted CTG reagent into each well of the cells, placing the cells on a shaking bed under the condition of room temperature and light shielding for mixing for 2 minutes to crack the cells, standing the cells for 10 minutes, and detecting the luminous intensity by using a microplate reader.

Crystal violet experiment: after the 6-well plate cells grew to a certain extent, the medium was decanted, washed twice with PBS, and fixed for 20min with 4% paraformaldehyde in each well. After repeated PBS washing twice, incubation was performed with crystal violet staining solution at room temperature for 30min in the dark. Washing with flowing water for several times, and taking pictures under an optical microscope for counting.

Soft agar experiments: after the 6-well plate cells grew to some extent, the medium was decanted, 1 XMTT staining solution was added, and the cells were placed in a 5% CO2 incubator. After 2h, the clones were stained bluish purple and photographed and scanned under an optical microscope.

Cell cycle detection: the cultured cells are prepared into single cell suspension, washed by PBS and fixed by 75 percent ethanol at the temperature of minus 20 ℃, washed by PBS after 24 hours, added with RNase A, evenly mixed and incubated at the temperature of 37 ℃ for 30 minutes, then added with Propidium Iodide (PI) to stain DNA, incubated at room temperature for 30 minutes in a dark place and detected by a flow cytometer.

Nude mouse xenograft experiments: the obtained GIST cells and matrigel are uniformly mixed according to the proportion of 1:1, and then the injection syringe is used for carrying out subcutaneous inoculation on axilla of a BALB/c nude mouse. The physiological condition of the mice was observed every day, and the tumor size was weighed. For drug-treated mice, after tumors had grown to some extent, imatinib was dosed twice daily at 50mg/kg and RO-3306 at 4mg/kg every two days, by oral gavage for 30 days. After 30 days of treatment, all mice were sacrificed and tumors were collected. Tumor volume and tumor weight were used to assess antitumor activity.

SA-beta-Gal cell senescence test experiment: after the lentivirus-infected cells were cultured until an obvious cell senescence state was observed under a microscope, the medium in the well plate was discarded, washed twice with PBS, and fixed with 4% paraformaldehyde for 20 min. Washed twice with running water, and incubated overnight with a Biyuntian beta-galactosidase activity assay kit. Discarding staining reagent, placing under an optical microscope for observation and photographing, taking pictures with 3 random visual fields, manually counting stained cells, and counting aging percentage.

The experimental steps are as follows:

1) constructing a CDK1 gene lentivirus knock-down vector;

2) infecting GIST430/654 and GIST-T1 cells after packaging lentivirus;

3) cell viability was measured using CellTiter-glo (CTG) kit (FIGS. 3A-B);

4) immunoblot detection of CDK1 knockdown levels (fig. 4H);

5) CDK1 was tested for its proliferative capacity by crystal violet, soft agar assay (fig. 3C-D);

6) flow cytometry examined the proportion of each phase of the cell cycle, which in turn reflected cell proliferation (fig. 3F);

7) injecting the cells into nude mice for xenotransplantation, and observing and detecting the growth of the tumor at regular intervals (FIG. 3E);

8) the cellular senescence kit detected the level of senescence in cells after CDK1 knockdown (fig. 3G).

The results of the experiment are shown in FIGS. 3 (A-G).

The results show that after the knock-down of CDK1, cell viability experiments, cell anchorage independent experiments, crystal violet experiments and nude mouse transplantation experiments all show that GIST cell proliferation is inhibited (FIG. 3, A-E); after knock-down of CDK1, the cell cycle was arrested at G0/G1 (FIG. 3, F) because of the increased cellular senescence (FIG. 3, G). Therefore, after CDK1 gene knockout, GIST cell growth and proliferation are obviously inhibited, and GIST senescence is promoted.

Example 4 CDK1 binds to and modulates phosphorylation of AKT

Vector construction: the full-length AKT and the FLAG-CDK1 are amplified through molecular cloning, and are connected into an expression vector through an enzyme digestion connection method.

Immunoprecipitation mass spectrometry coupled with: cells were lysed with IP buffer containing protease inhibitors (10. mu.g/mL leukin, 10. mu.g/mL aprotinin and 1mM phenylmethylsulfonyl fluoride), mixed overnight with 1. mu.g anti-FLAG antibody and 20. mu.L protein G-agarose (ThermoFisher #101242), and eluted by boiling with SDS loading buffer. The eluted samples were detected by SDS-PAGE, Coomassie Brilliant blue staining (colloidal blue staining kit, Invitrogen, # LC 6025). For mass spectrometry, IP samples were eluted by shaking with 8M urea and 100mM Tris-Cl (pH 8.0) and subjected to mass spectrometry.

Co-immunoprecipitation: cells were lysed with IP buffer containing protease inhibitors (10. mu.g/mL leupeptin, 10. mu.g/mL aprotinin and 1mM phenylmethylsulfonyl fluoride), mixed with 2. mu.g anti-Flag antibody and 20. mu.L protein G-agarose and incubated overnight. The immunoprecipitates were eluted by boiling with SDS loading buffer. IP samples and whole cell lysates were analyzed by western blot.

The experimental steps are as follows:

1) constructing a FLAG-CDK1 exogenous expression vector;

2) overexpression of FLAG-CDK1 in GIST430/654, pulling down of CDK1 interacting proteins using FLAG antibody, mass spectrometric identification (FIG. 4A);

3) overexpression of FLAG-CDK1 in GIST430/654, pull-down using FLAG antibody, validation of the interacting protein AKT (FIG. 4D);

4) treatment in GIST430/654 with the CDK1 inhibitor RO-3306, validated the CDK1 specific interacting protein AKT (FIG. 4D);

5) in vitro binding experiments, CDK1 inhibitor RO-3306 was incubated and pulled down with CDK1 antibody, validating CDK1 interacting protein AKT1 (fig. 4E);

6) FLAG-CDK1 and HA-AKT were introduced exogenously in the 293 system, and interaction between CDK1 and AKT was verified by using FLAG antibody pull-down (FIG. 4F);

7) exogenous FLAG-CDK1 was introduced into PDK1 normal and deficient GIST430/654 cells, and CDK1 was verified to interact with AKT independent of PDK1 by FLAG antibody pull-down (fig. 4B);

8) in PDK1 normal GIST430/654, cells were treated with the AKT inhibitor MK-2206, pulled down with FLAG antibody, verifying CDK1 binding to AKT, immunoblotted with whole cell lysates, showing that inhibitor treated groups reduced phosphorylation of AKT (fig. 4C);

9) in PDK1 knockout GIST430/654 cells, FLAG-CDK1 and HA-AKT1 were exogenously introduced, and the interaction protein AKT was verified by FLAG pull-down, which was shown by immunoblotting to reduce the phosphorylation of AKT (FIG. 4G);

10) the expression levels of AKT/pAKT, PCNA and PARP, etc., were examined by Western blotting using CDK1 inhibitor RO-3306 or knocking-down CDK1 in GIST430/654 and GIST-T1 (FIG. 4H-I);

the results of the experiment are shown in FIG. 4 (A-I).

The results showed that AKT1 ranked second among the proteins pulled down using antibody FLAG in GIST430/654 (FIG. 4A); the binding of CDK1 and AKT was independent of PDK1 (fig. 4B); treatment with MK-2206, an inhibitor of AKT, inhibited CDK1 binding to AKT and decreased the level of phosphorylation of AKT in PDK1 normal GIST430/654 cells (fig. 4C); AKT1 interacted with CDK1 in GIST cell lines (fig. 4D); in vitro interaction assays also showed that CDK1 interacted with AKT (fig. 4E); in the 293 cell system, AKT1 interacted with CDK1, and CDK1 promoted phosphorylation of threonine 308 and serine 473 of AKT (fig. 4F); knock-down of CDK1 in GIST430/654 and GIST-T1 cells inhibited AKT phosphorylation, induced apoptosis, reduced PCNA expression (FIG. 4G, H); treatment of GIST430/654 and GIST-T1 cells with RO-3306 inhibited AKT phosphorylation, induced apoptosis, and inhibited cell proliferation as did CDK1 knockdown (FIG. 4I). Thus, CDK1 binds and modulates phosphorylation of AKT and is PDK1 independent, while CDK1 knockdown promotes apoptosis.

Example 5 CDK1 promotion of GIST growth and proliferation by AKT

Co-immunoprecipitation: cells were lysed with IP buffer containing protease inhibitors (10. mu.g/mL leupeptin, 10. mu.g/mL aprotinin and 1mM phenylmethylsulfonyl fluoride), mixed with 2. mu.g anti-Flag antibody and 20. mu.L protein G-agarose and incubated overnight. Immunoprecipitates were eluted by boiling with SDS loading buffer. IP samples and whole cell lysates were analyzed by western blot.

The experimental steps are as follows:

1) AKT was replenished after knocking-down CDK1 in GIST430/654, and GIST growth and proliferation were examined by CellTiter-Glo viability, crystal violet, and soft agar experiments (FIGS. 5A-C);

2) AKT was restored after CDK1 knock-down in GIST430/654, and GIST growth and proliferation were examined by in vivo experiments such as nude mouse transplantation (FIG. 5D).

The results show that supplementing AKT complements the phenotype of cell viability inhibition after CDK1 knockdown (fig. 5A); complementation of AKT, complemented the phenotype of inhibition of cell proliferation after CDK1 knockdown (fig. 5B); complementation of AKT, complementing a phenotype independent of inhibition of cell anchorage after CDK1 knockdown (fig. 5C); AKT was recruited to compensate for CDK1 knockdown and inhibited tumor growth in xenograft mice (fig. 5D). Thus, CDK1 promotes GIST growth and proliferation through AKT.

Example 6 anti-tumor Activity of CDK1 inhibitors on Imatinib-resistant and Imatinib-sensitive advanced GIST

The experimental steps are as follows:

1) 7 GIST cells and GIST primary cells were treated with gradient RO-3306, cell viability was measured with CellTiter-Glo viability kit and half inhibitory concentrations were counted (FIGS. 6A-B);

2) CDK1 expression levels in GIST cells as well as GIST primary cells were shown by immunoblotting (fig. 6C);

3) GIST cells as well as primary GIST cells were treated with RO-3306 and immunoblots showed decreased AKT phosphorylation (FIG. 6D);

4) in GIST-T1 cell line, treatment with RO-3306 in combination with imatinib was significantly superior to monotherapy (FIG. 6H);

5) tumor size, volume and toxic effects on mice were examined for patient-derived xenograft mice (PDX) using RO-3306 or a combination of RO-3306 and imatinib (FIG. 6E-G, I-K).

The results show that RO-3306 inhibited imatinib-sensitive and resistant GIST cell lines, without affecting GIST primary cells and fibroblasts that do not express CDK1 (fig. 6, a-B); expression level of CDK1 in GIST cells as well as GIST primary cells (fig. 6C); treatment of imatinib-resistant and imatinib-sensitive GIST cells with RO-3306 did not affect the expression of AKT, but significantly reduced the phosphorylation level of AKT (fig. 6D); the CDK1 inhibitor inhibited tumor volume and size in imatinib-resistant GIST xenograft mice, with no toxic effects (fig. 6, E-G); treatment of imatinib-sensitive GIST-T1 tumor-bearing mice with a CDK1 inhibitor in combination with imatinib reduced tumor volume as well as tumor size. And CDK1 inhibitors did not affect the body weight changes of imatinib-sensitive GIST xenograft mice (fig. 6, I-K). Thus, CDK1 inhibitors have anti-tumor activity against imatinib-resistant and imatinib-sensitive advanced GIST.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a CDK1 gene, mRNA, cDNA, or protein, or a detection reagent therefor, (i) as a marker for the detection of advanced gastrointestinal stromal tumors; and/or (ii) for preparing a diagnostic reagent or kit for detecting advanced gastrointestinal stromal tumors.

2. A diagnostic kit for detecting advanced gastrointestinal stromal tumors, comprising a container comprising a detection reagent for CDK1 gene, mRNA, cDNA, or protein; and a label or instructions indicating that the kit is for detecting advanced gastrointestinal stromal tumors.

3. A method of detecting advanced gastrointestinal stromal tumors, the method comprising:

a) providing a test sample from a subject;

b) detecting the expression level of CDK1 protein in the test sample E1; and

wherein an expression level of CDK1 protein in the sample above a reference value indicates that the subject has advanced gastrointestinal stromal tumor.

4. A method of determining a treatment plan, comprising:

a) providing a test sample from a subject;

b) detecting the expression level of CDK1 protein in the test sample; and

5. Use of an inhibitor of the CDK1 gene or protein thereof, for the preparation of a composition or formulation for (a) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (b) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (c) increasing the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

6. A pharmaceutical composition, comprising:

(a1) an inhibitor of the CDK1 gene or protein thereof;

(a2) tyrosine kinase inhibitors; and

(b) a pharmaceutically acceptable carrier.

7. A product combination, comprising:

8. A kit, comprising:

9. Use of a pharmaceutical composition according to claim 6 or a product combination according to claim 7 or a kit according to claim 8 for the preparation of a medicament for (i) inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells; and/or (ii) preventing and/or treating advanced gastrointestinal stromal tumors; and/or (iii) agents that increase the resistance and sensitivity of advanced gastrointestinal stromal tumor cells to tyrosine kinase inhibitors.

10. An in vitro non-therapeutic method of inhibiting the growth or proliferation of advanced gastrointestinal stromal tumor cells, comprising the steps of: culturing advanced gastrointestinal stromal tumor cells in the presence of a CDK1 gene or a protein inhibitor thereof, thereby inhibiting growth or proliferation of gastrointestinal stromal tumor cells.

11. A method of screening for a candidate compound for the prevention and/or treatment of advanced gastrointestinal stromal tumors, the method comprising the steps of:

(a) in the test group, a test compound is added to a culture system of cells, and the expression amount (E1) and/or activity (a1) of CDK1 is observed in the cells of the test group; in a control group, no test compound was added to the culture system of the same cells, and the expression amount (E0) and/or activity (a0) of CDK1 in the cells of the control group was observed;